Molecular and Cellular Biochemistry 121: 59-65, 1993. 9 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Verapamil induced reduction of the myocardial -adrenoceptor density in BIO 14.6 cardiomyopathic Syrian hamsters Akira Kobayashi, Terumasa Nishiyama, Takayoshi Ikegaya, Masanori Kaneko, Noboru Yamazaki The Third Departmentof Internal Medicine, Hamamatsu UniversitySchool of Medicine, 3600, Handa-cho, Hamamatsu, Japan, 431-31 Received 7 August 1992; accepted 30 November 1992

Abstract In general, it is recognized that prolonged exposure to catecholamine leads to a reduction in the 6-adrenoceptor density (downregulation). However, it has been previously reported that the myocardial [3-adrenoceptor densities and norepinephrine levels significantly increase in the hearts of BIO 14.6 cardiomyopathic hamsters in the early stage. The mechanism of the increased ]3-adrenoceptor density is not clearly elucidated, and it can not be excluded that this phenomenon may be a secondary effect. The purpose of this study was to assess the effect of verapamil on the density of 13-adrenoceptors in the heart of BIO 14.6 cardiomyopathic hamsters. The total number of {3-adrenoceptors in untreated BIO 14.6 hamsters was significantly higher at 90 days of age (30.4+ 2.2 v.s. 25.9+ 1.4 fmol/mg protein, p < 0.05). BIO 14.6 hamsters received daily intraperitoneal injections of 5 mg/kg verapamil for 70 days, from an age of 20 days. Verapamil protected against progressive myocardial damage (total damage; 8.2+ 0.7 v.s. 0.4+ 0.2%/area, p < 0.05) and the myocardial [3-adrenoceptor density returned to that of the normal control group (26.9+ 3.0fmol/mg protein). Conversely, verapamil did not have an effect on the number of myocardial ]3-adrenoceptors in normal golden hamsters. This study showed that verapamil protected against progressive myocardial damage and myocardial [3-adrenoceptor density returned to those of normal hamsters. These results suggest that an increased number of [3-adrenoceptors in the early stage of BIO 14.6 cardiomyopathic hamsters may be involved in the secondary pathogenesis of cardiomyopathy. (Mol Cell Biochem 121: 59-65, 1993)

Key words."cardiomyopathy, upregulation, downregulation, norepinephrine, ]3-adrenoceptor density

Introduction The number of receptors per unit area of the sarcolemma (the receptor density) is not a fixed quantity but can change in response to certain physiological or pathophy-

siological circumstances [1, 2]. These changes are called upregulation and downregulation, indicating the direction of the response. In general, it is well known that pro-

Address for offprints." A. Kobayashi, The Third Department of Internal Medicine, Hamamatsu University School of Medicine, 3600, Handa-cho, Hamamatsu, Japan, 431-31

60 longed catecholamine exposure of myocytes leads to a reduction in the number of myocardial [3-adrenoceptors (downregulation). This phenomenon, however, does not appear in the early stage of the cardiomyopathic heart of BIO 14.6 Syrian hamsters. Many investigators reported that both ~3-adrenoceptor density and catecholamine levels significantly increased in the heart of BIO 14.6 cardiomyopathic hamsters, and that sympathetic nervous system abnormalities may contribute to the pathogenesis and development of cardiomyopathy [3-51. On the other hand, an increase in myocardial [3-adrenoceptor density was not observed at 21 days of life in hamsters which had no cardiac abnormalities [6]. Thus, the increased [3-adrenoceptor density is absent before the development of cardiomyopathy and is not related to a genetic lesion. The mechanism of the increased 13-adrenoceptor density is not clearly elucidated, and it can not be excluded that this phenomenon may be a secondary effect of the heart disease. If this theory is true, the increased density of myocardial [3-adrenoceptors may be decreased when the myocardial damage is improved by administration of verapamil, which has been the most effective drug administered to these types of hamsters [8]. Therefore, this study was performed to elucidate whether or not verapamil treatment prevented the increased [3-adrenoceptor density in BIO 14.6 hamsters.

Methods Animals The BIO 14.6 cardiomyopathic hamsters used in this study were purchased from the Central Institute for Experimental Animals (Japan) and bred in the Experimental Animal Center of Hamamatsu University School of Medicine. Age-matched golden healthy hamsters were purchased from the Inoue Experimental Animal Center (Japan). All experiments conformed to the Hamamatsu University School of Medicine regulations governing the care and use of laboratory animals.

Treatment protocol Twenty-four 20-day of old BIO 14.6 cardiomyopathic hamsters were divided into two groups: a verapamil-

treated group (n = 12) and a non-treated group (n = 12). Fourteen age-matched golden healthy hamsters were also divided into two groups, as with the BIO 14.6 hamsters. Verapamil-treated animals received intraperitoneal injection of 5 mg/kg verapamil for 70 days from an age of 20 days. Non-treated groups received similar volumes of saline solution for the same duration as the verapamil-treated groups. Of the 24 BIO 14.6 cardiomyopathic and 14 golden healthy hamsters entered into the present study, one verapamil-treated BIO 14.6 hamster died during treatment with verapamil. All of the animals were killed by cervical dislocation at 90 days of life, 30hr after the last dosage of verapamil.

Histological study BIO 14.6 cardiomyopathic hamsters were used in the histological study [verapamil-treated group (n = 5), nontreated group (n = 4)]. The hearts were sliced at the center into pieces 5 mm thick, which were immediately fixed in 10 % buffered formalin. After 24-48 hr of fixation, the sections were embedded in solid paraffin. The pieces of tissue were sliced into sections 3gm thick and stained with haematoxylin and eosin, Mallory-Heidenhain triples stain, and yon Kossa's stain. The tissue specimens were photographed at 60x magnification after each stain. The total area of heart muscle, as well as the area of necrosis, fibrosis, and calcification in the heart muscle tissue specimens, were traced from the photographs and measured using a image analyser system, Video Plan (Kontron, Germany). The ratios of the necrosis, fibrosis, and calcification areas to the total area of the whole heart muscle section were compared for the verapamiltreated group and the non-treated group.

Cardiac tissue preparation The heart of each was trimmed of the atrium and large vessels, with the right and left ventricles cut into small pieces. These pieces were homogenized 3 times for 15 seconds each time with a polytron in 50mM Tris/HC1 buffer (pH7.4). The homogenates were further treated with a Potter-Elvehjem homogenizer.The homogenates were centrifuged at 39,000x g (maximum) for 20min at 4 ~C. The pellets were washed twice, then resuspended

61 total damage

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in fresh 50mM Tris/HC1 buffer. The final pellets were resuspended to a concentration of 100mg original wet tissue weight per ml buffer.

If a significant F resulted from ANOVA (p< 0.05), the Duncan's multiple range test was applied. The histological changes were compared using the unpaired Student's t-test. A p value of less than 0.05 was considered a significant change.

Cardiac ~-adrenoceptor assays Aliquots (50gl) of cardiac membrane homogenates were incubated with six different concentrations of cyanopindolol ([125I]CYR 0.003 to 0.25nM) containing incubation buffer (pH7.4) with or without 10pM propranolol. The incubations were carried out for 90min at 30~ Membrane bound [125I]CYP was trapped at the end of the incubation period by rapid vacuum filtration of the mixture over a Whatman glass fiber filter. The filter was rinsed and radioactivity trapped on the filter was measured. Nonspecific binding was defined as the binding of [125I]CYP in the presence of 10gM propranolol and was subtracted from the total to yield the specific binding assay. Specific binding of [~25I]CYP at Kd was in the range of 80-90% of the total. The binding assay was duplicated. Protein concentration was measured using the method of Lowry et al. [7].

Results Effect of verapamil on histology in BIO 14.6 hamsters The total area and the percent area damage in the verapamil-treated group were significantly smaller than those in the non-treated group (11.4+ 6.3 v.s. 192.0+ 33.7 10 2ram2 and 0.44_+ 0.23 v.s. 8.18_+ 0.66%, both p< 0.01) Fig. 1. The areas and percent areas of fibrosis and calcification in the verapamil-treated group were significantly smaller than those in the non-treated group (fibrosis; 4.7+ 2.8 v.s. 104.2+ 9.4 10-2mm 2 and 0.17+ 0.11 v.s. 4.63+ 0.56%, both p < 0.05: calcification; 2.2+ 2.2 v.s. 45.2+ 14.7 10-2mm 2 and 0.09_+ 0.09 v.s. 1.90_+ 0.50%, both p< 0.05). These results show that early verapamil treatment can protect against early myocardial damage in BIO 14.6 cardiomyopathic hamsters.

Statistical analys& Data obtained in the [3-adrenoceptor binding study and in the histological study were represented as the mean +S.D. The statistical significances of the differences of [3-adrenoceptor binding between verapamil-treated and non-treated groups were determined by using ANOVA.

Cardiac ~-adrenoceptor binding sites in BIO 14.6 hamsters The results of equilibrium binding measurements using 6 different concentrations of [125I]CYP showed that [~25I]CYP binds in a saturable manner to homogenates

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Fig. 2. Equilibrium Binding of [tzsI]CYPand Scatchard Plot of [125"I]CYP Binding. A typical saturation experiment of [12:~5I]CYPbindinginthehamster ventricle. Samples of the tissue preparation were incubated with varying concentrations of [t25I]CYP(0.003-0.25 nM) for 90 rain at 30~C. The concentration-dependent specific [125I]CYPbinding is shown on the left and the Scatchard plot of the specific binding is shown on the right.

of the hamster's ventricle (Fig. 2). Each Scatchard plot was linear indicating a consistent mass action behavior with respect to the binding of [125I]CYRThe dissociation constant (Kd) and maximum binding (Bmax) were determined from the Scatchard plots for each hamster ventricle. The total number of [~-adrenoceptors in BIO 14.6 cardiomyopathic hamsters was significantly increased compared to golden healthy hamsters at 90 days of life (30.4+ 2.2 v.s. 25.9+ 1.4 fmol/mg protein, p< 0.05, Fig. 3). On the other hand, there was no significant difference in the [3-adrenoceptor affinity (Kd) between BIO 14.6 cardiomyopathic hamsters and golden healthy hamsters (19.5+ 5.4 and 18.1+_5.2pM, respectively).

Effect of verapamil on myocardial [5-adrenoceptors in golden healthy hamsters The total number of [3-adrenoceptor in verapamil-treated and non-treated golden healthy hamsters was 26.1+ 1.7 and 25.9+. 1.4fmol/mg protein, respectively, with no significant difference between them (Fig. 4). Similarly, there was no significant difference in ~3-adren0ceptor affinity (Kd) between verapamil-treated and non-treated golden healthy hamsters (17.6+ 2.2 and 18.1+ 5.2pM, respectively).

Effect of verapamil on myocardial ~-adrenoceptors in BIO 14.6 hamsters. The total number of myocardial [3-adrenoceptors in verapamil-treated and non-treated BIO 14.6 cardiomyopathic hamsters was 26.9+ 3.0 and 30.4+ 2.2fmol/mg protein, respectively, with significant difference between them (p< 0.05, Fig. 3). On the other hand, there was no significant difference in myocardial 13-adrenoceptor affinity (Kd) between verapamil-treated and non-treated BIO 14.6 cardiomyopathic hamsters (15.9+ 3.5 and 19.5+ 5.4pM, respectively).

Discussion Verapamil has been the most effective drug administered to BIO 14.6 cardiomyopathic Syrian hamsters to date [8]. The major effect of verapamil is to suppress the movement of calcium ions through calcium ion selective, voltage-dependent channels. Several recent reports have indicated that calcium antagonist reagents may be cell membrane receptor blockers [9-11]. Hedberg et al. have demonstrated that the [3-adrenoceptor density is increased after the administration of a calcium antagonist [9]. On the other hand, Karliner et al. and Nayler et al. have reported that verapamil is a competitive antagonist of myocardial cr but not

63

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BIO 14.S Fig. 3. Cardiac [3-Receptor Binding Sites at 90 days. Values are mean +S.D. for each group. The significance of the changes are as follows: G.H.H. vs.VER(-), VER(-) vs. VER(+), *p <0.05. G.H.H.~ golden healthy hamster; VER(-) = BIO 14.6 hamster without verapamil treatment; VER(+)= verapamil-treated BIO 14.6 hamster.

VER.(*) G.H.H.

Fig. 4. Effect of verapamil on Cardiac {3-Receptor Binding Sites in Normal Heart. Values are mean + S.D. for each group. The significance of the changes are as follow: VER(-) vs. VER(+). V E R ( - ) = group without verapamil treatment; VER(+)= verapamil-treated group; G.H.H. = golden healthy hamster. et al. demonstrated that myocardial catecholamine lev-

of [3-adrenoceptor [10, 11]. In this study, we investigated the effect of verapamil on the number of myocardial [3-adrenoceptors in normal golden hamsters and obtained a result that the verapamil did not produce a significant change. The cardiac disease of BIO 14.6 cardiomyopathic hamsters can be divided into four phases [12]. During the first phase (prenecrotic phase), the animals appear healthy and there is no pathologic evidence of heart disease. The second phase begins when the animals are about 30 days old, and is characterized by the appearance of focal myocardial necrotic lesions. At about 90 to 120 days of age, many of the necrotic lesions have healed, few new lesions appear, and hypertrophy of the heart begins. The terminal phase is marked by cardiac dilatation and overt congestive heart failure. We previously reported that the number of myocardial [~-adrenoceptors was not elevated at 21 days of age, but increased in the second (40 days old) and hypertrophic phases (90 days old) [6]. On the other hand, Sole

els were already increased from the prenecrotic phase [4]. We investigated the changes of myocardial [3-adrenoceptor density in the early stage of cardiomyopathy (from 20 days to 90 days of age) in the BIO 14.6 cardiomyopathic hamsters in this study. This study showed that verapamil protected against progressive myocardial damage and of myocardial J3-adrenoceptor density returned to that of golden healthy hamsters. This normalization of myocardial [3-adrenoceptor density was not due to a direct effect of verapamil on myocardial [3-adrenoceptors, because verapamil did not affect the number of myocardial [3-adrenoceptors in golden healthy hamsters. In addition, it is necessary to investigate the possible mechanisms for the reduction in the number of myocardial [3-adrenoceptors in BIO 14.6 hamsters after treatment with verapamil. We can not explain the mechanisms from this study, but there are many possible mechanisms for it. One possible mechanism is a response to change in the activity of the adenylate cyclase-G protein

64 system. Kessler et aL [13] and Ikegaya et al. [14] demonstrated functional defects in the cardiac Gs protein and adenylate cyclase activities reduced in BIO 14.6 hamsters. Since reduced Gs protein bioactivity could induced abnormal myocardial contractility [15], an increased [3-adrenoceptor density may contribute to maintenance of the normal myocardial function in the early stage of cardiomyopathy. However, the diminished Gs protein bioactivity has been shown to be present before the development of cardiomyopathy (at 29-day-old) [13], so there is little possibility of the defect in the activity of the adenylate cyclase-G protein system. Another possible mechanism is free radical injury. Free radicals affect membrane lipids, and lipid peroxide clusters and changes in membrane fluidity [16]. Cell membrane fiuidity is an inherent property of membrane, the alteration of which affects the accessibility of surface receptors to ligand binding [17,18]. Kaneko et al. reported that the maximal number, B .... of ~-adrenoceptor was increased by xanthine plus xanthine oxidase [19]. Kobayashi et aL demonstrated that the concentration of lipid peroxide and free radicals in the heart were significantly higher in BIO 14.6 hamsters [6]. Sakanashi et al. reported that lipid peroxide concentrations were significantly higher in the hearts of BIO 14.6 hamsters at 60 days of age, and that vitamine E administration effectively restored creatinine-kinase activity and decreased the lipid peroxide content in the myocardium [20]. Kobayashi et al. demonstrated that verapamil inhibited the increase in free radical concentration in the heart of BIO 14.6 hamsters, returning this to a normal control level [6]. These results suggest that free radical injury may increase the membrane fluidity and concurrently increase the ~-adrenoceptor density, and that histological improvement may be possible mechanism for the reduction of ~-adrenoceptor density in BIO 14.6 hamster after treatment with verapamil. We also previously examined the regional variation in [3-adrenoceptor density within the ventricles in BIO 14.6 hamsters [21]. The [3-adrenoceptor density was significantly increased, corresponding to the sites of increased interstitial fibrosis. This observation indicates that the supersensitivity phenomenon could contribute to the development of degenerative changes in cardiomyopathy in the BIO 14.6 hamsters. In conclusion, the increased myocardial ~-adrenoceptot density was significantly reduced in BIO 14.6 hamsters which were prevented the pathological damage of cardiomyopathy after treatment with verapamil. These results suggest that an increase in the myocardial [~-adre-

noceptor density in the early stage of BIO 14.6 cardiomyopathic hamsters may be involved in the secondary pathogenesis of cardiomyopathy. However, an increased [3-adrenoceptors density could be implicated in the later progression of cardiomyopathy in the BIO 14.6 hamster.

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